Microbial Cell Factories BioMed CentralLdh(s) under the control of the same ScTPI promoter (data not shown). Improving lactate production and yield by protein engineering The Ldh enzyme

BioMed CentralMicrobial Cell Factories

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Open AcceResearchLactate production yield from engineered yeasts is dependent from the host background, the lactate dehydrogenase source and the lactate exportPaola Branduardi1, Michael Sauer2,3, Luca De Gioia1, Giuseppe Zampella1, Minoska Valli2,3, Diethard Mattanovich2,3 and Danilo Porro*1
Address: 1Università degli Studi di Milano – Bicocca, Dipartimento di Biotecnologie e Bioscienze, P.zza della Scienza 2, 20126 Milano, Italy, 2Institute of Applied Microbiology, BOKU – University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Wien, Austria and 3Fh-campus wien – University of Applied Sciences, School of Bioengineering, Muthgasse 18, A-1190 Wien, Austria

Email: Paola Branduardi - [email protected]; Michael Sauer - [email protected]; Luca De Gioia - [email protected]; Giuseppe Zampella - [email protected]; Minoska Valli - [email protected]; Diethard Mattanovich - [email protected]; Danilo Porro* - [email protected]

* Corresponding author

AbstractBackground: Metabolic pathway manipulation for improving the properties and the productivityof microorganisms is becoming a well established concept. For the production of importantmetabolites, but also for a better understanding of the fundamentals of cell biology, detailed studiesare required. In this work we analysed the lactate production from metabolic engineeredSaccharomyces cerevisiae cells expressing a heterologous lactate dehydrogenase (LDH) gene. TheLDH gene expression in a budding yeast cell introduces a novel and alternative pathway for theNAD+ regeneration, allowing a direct reduction of the intracellular pyruvate to lactate, leading toa simultaneous accumulation of lactate and ethanol.

Results: Four different S. cerevisiae strains were transformed with six different wild type and onemutagenised LDH genes, in combination or not with the over-expression of a lactate transporter.The resulting yield values (grams of lactate produced per grams of glucose consumed) varied fromas low as 0,0008 to as high as 0.52 g g-1. In this respect, and to the best of our knowledge, higherredirections of the glycolysis flux have never been obtained before without any disruption and/orlimitation of the competing biochemical pathways.

Conclusion: In the present work it is shown that the redirection of the pathway towards thelactate production can be strongly modulated by the genetic background of the host cell, by thesource of the heterologous Ldh enzyme, by improving its biochemical properties as well as bymodulating the export of lactate in the culture media.

BackgroundMetabolic engineering can be defined as the directedimprovement of product formation or cellular properties

through the modification of specific biochemical reac-tions or introduction of new ones with the use of recom-binant DNA technology. Aimed to produce single

Published: 30 January 2006

Microbial Cell Factories 2006, 5:4 doi:10.1186/1475-2859-5-4

Received: 28 November 2005Accepted: 30 January 2006

This article is available from: http://www.microbialcellfactories.com/content/5/1/4

© 2006 Branduardi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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compounds, metabolic engineering necessarily includesthe modification of the cellular pathway(s) as well as theredirection of the energy toward the production itself (seefor a review [1]). The existing metabolic engineeringapplications are the result of more than two decades ofglobal experience developing processes for the productionof fine chemicals, vitamins, nutraceuticals and animalnutritional supplements such as amino acids (as exam-ples, see [2-5]). Given their relatively low complexity, thefirst biotechnological applications have been developedin microorganisms.

Different research teams have been involved in the pro-duction of lactate from metabolic engineered yeasts suchas Saccharomyces cerevisiae [6-13], Kluyveromyces lactis[14,15], Torulaspora delbrueckii [16] and Zygosaccharomycesbailii [17]. L-Lactic acid, first discovered by the Swedishchemist Scheele (1780), has been traditionally used as afood preservative and food flavoring compound [18].Recently, it has received attention since it can be used toproduce a biodegradable polymer with plastic properties.The market for this organic acid is rapidly growing,exceeding several hundred million dollars annually [10].This carboxylic acid is currently mainly produced usinglactic-acid bacteria, such as various Lactobacillus spe-cies,(via an, anaerobic fermentation that operates opti-mally at pH values where the salt of the organic acid ratherthan the free acid is formed, although free lactic acid ispreferred for most industrial processes [18]. The use ofmicroorganisms like yeasts that are more tolerant to lowpH values than the current production organisms, couldstrongly decrease the amount of neutralizing agentsrequired and lower the cost of down-stream processing.Pyruvate is the end product of glycolysis; it can be furthermetabolized either by the pyruvate dehydrogenase com-plex (Pdh, EC 1.2.4.1) to acetyl-coenzyme A or by pyru-vate decarboxylase (Pdc, EC4.1.1.1) to acetaldehyde and

subsequently to ethanol. In previous works it has beenshown that the expression of a heterologous lactate dehy-drogenase (Ldh, EC 1.1.1.27) gene in the above men-tioned yeast hosts introduces a new and alternativepathway for the NAD+ regeneration, allowing a directreduction of the intracellular pyruvate to lactate leading toa simultaneous formation of ethanol and lactic acid [7].Only following a partial or a full replacement of the etha-nol production by the lactate production, obtained inrecombinant yeast cells lacking the Pdc and/or Pdh activ-ities, it has been possible to obtain lactate productionwith high yield (max reported yield: 0.85, i.e., gram of lac-tate produced per gram of glucose consumed) values [15].Theoretically, 2 moles of lactate and 2 moles of ATP areformed per mole of glucose consumed. In the presentwork we show that in S. cerevisiae cells, the yield value canbe strongly modulated by the genetic background of thehost, by the source of the heterologous Ldh enzymes, byimproving their biochemical properties as well as by mod-ulating the export of lactate in the culture media. Follow-ing all these approaches we have been able to improve theyield from values as low as 0,0008 to values as high as0.52 without any modulation of the Pdc and/or Pdh activ-ities.

Results and discussionExpression of different LDH(s) in different yeast hostsLactic acid has been already produced by metabolicallyengineered yeast hosts (see Introduction). To betterunderstanding the modulation of lactate production fromthe background of wild type S. cerevisiae cells, we firstlytested lactate production from different S. cerevisiae yeaststrains transformed with the same integrative plasmid,pB1. As a model LDH gene we chose the mammalian Ldh-A lactate dehydrogenase. The transcription of the heterol-ogous gene is under the control of the strong constitutiveS. cerevisiae TPI (Triose Phosphate Isomerase) promoter.

Table 1: Plasmids (all integrative) utilized in this study*

Vector Promoter Heterologous protein Selection marker Reference

pB1 ScTPI BtLDH URA3 this studypB2 ScTPI BtLDH HIS3 this studypB3 ScTPI BtLDH LEU2 this studypBME2 ScTPI BmLDH URA3 this studypBST2 ScTPI BsLDH URA3 this studypLC5 ScTPI LcLDH URA3 Brambilla et al., 1999pLC7 ScTPI LcLDH HIS3 Brambilla et al., 1999p022TLP ScTPI LpLDH HIS3 this studyp012TLP ScTPI LpLDH URA3 this studyp022TLPD94G ScTPI LpLDHD94G HIS3 this studyp012TLPD94G ScTPI LpLDHD94G URA3 this studyp012Jen1 ScTPI ScJen1 URA3 this studyp022Jen1 ScTPI ScJen1 HIS3 this study

*a complete description of plasmids construction is given in Materials and MethodsAbbreviations: Sc: S. cerevisiae; Bt: Bos taurus; Bm: Bacillus megaterium Bs: Bacillus stearothermophylus; Lc: Lactobacillus casei; Lp: Lactobacillus plantarum



The resulting expression vector has been used to trans-form five different S. cerevisiae host strains. Transformedstrains have been grown in batch shake-flask culture on2% wv-1 glucose-YNB based media. Further, to betterunderstand the modulation of the production from differ-ent Ldh(s), we also tested the lactate production from thesame S. cerevisiae yeast strain (GRF18U) transformed withthe same integrative expression vector, but bearing differ-ent Ldh(s).

Table 2 compares the lactate and the ethanol productionsobtained. Independently from the yeast strain used and ofthe heterologous enzyme, the highest accumulation oflactate and ethanol were observed at the beginning of thestationary phase (reached, in these growth conditions,after about 30 hours from the inoculum), in coincidencewith the exhaustion of glucose (data not shown; see forexample also figure 1A and 1B). Findings reported indi-cate that the lactate production is strongly dependentfrom the background of the hosts. Furthermore, a higherlactate production comes along with a lower ethanol accu-mulation (data not shown). Productions ranging from 20to 801 mg l-1 have been obtained from different yeasthosts expressing one copy of the same bovine Ldh andyielding similar specific activities (ranging from 0,4 to 0,6U mg-1). If two or three gene copies are expressed, theproduct levels (up to 1110 mg l-1) and activity (up to 0,8U/mg) increase, even if the improvement not propor-tional to the gene copy number. Data reported in theTable 2 further indicate that different Ldh(s) lead to differ-ent lactate productions (from 20 to 6150 mg l-1). On one

side it is interesting to underline that, despite the high Kmvalue for pyruvate, high productions (4160 mg l-1) havebeen observed transforming the GRF18U yeast host withthe Lactobacillus casei Ldh gene. On the other side, lactateconcentrations of the same order (6150 mg l-1) have beenobtained using the fructose1-6BP independent L.plantarum Ldh, which has a much lower Km value (Table2).

In general, and as expected, it can be concluded that thehigher the specific activity is, the more efficient is the con-version of pyruvate to lactate.

Similar results have been obtained transforming the hostcells with identical centromeric plasmids bearing differentLdh(s) under the control of the same ScTPI promoter(data not shown).

Improving lactate production and yield by protein engineeringThe Ldh enzyme is composed of four identical subunits[19]. All the bacterial Ldh enzymes listed in Table 2, withthe exception of the L. plantarum Ldh, require fructose 1-6BP as a cofactor. It has been proposed that the rate limit-ing step of the enzymatic reaction corresponds to the con-formational rearrangement of the so-called catalytic loop[20]. In particular, the movement of the catalytic loop isnecessary to allow pyruvate binding and L-lactate release.

Looking at a computational development of a better Ldhenzyme, we initially took into consideration the spiny

Table 2: Lactate and ethanol productions from different S. cerevisiae hosts transformed with different Ldh(s).

plasmid LDH Lactate EtOH Yield LDH activity LDH Km

Strains (source) mg l-1 mg l-1 g g-1 U mg-1 mM

W303-1A pB1 B. taurus 20 6430 0,0010 0,4 1,00 [19]CEN.PK pB1 B. taurus 140 6340 0,0070 0,5 1,00GRF18U pB1 B. taurus 801 5750 0,0401 0,6 1,00MB11 pLC5 L. casei 200 6110 0,0100 Nd 10,00 [19]GRF18U pB1; pB2 B. taurus 950 5730 0,0475 0,7 1,00GRF18U pB1; pB2; pB3 B. taurus 1100 5648 0,0550 0,8 1,00GRF18U pBST2 B. stearotherm. 128 6310 0,0064 0,6 0,03 [19]GRF18U pBME2 B. megaterium 1371 5672 0,0686 0,7 NdGRF18U pLC5 L. casei 4160 4270 0,2080 5,5 10,00GRF18U p022TLP L. plantarum 6150 3730 0,3075 3,2 1,50 (this study)

All the indicated recombinant yeast strains, together with the respective control here not reported, have been grown in shake-flasks on 2% (w/v) glucose-YNB based media till stationary phase, reached after about 30 hours after the inoculum. Cultures were independently repeated, after the screening of independent transformants, (at least) three times, rising to comparable results. In the table one fermentation per each set of experiment is reported, being the independent kinetics reproducible among themselves.Lactate and ethanol production (mg l-1, columns 4 and 5, respectively) correspond to the highest measured production levels.The Yield value (column 6) represents the grams of lactate produced per gram of glucose consumed (max theoretical yield = 1).Ldh activity (column 7) has been determined at the highest lactate production level.Km values against NADH are comprised between 0.001 and 0.0045 mM [19].Km apparent value of mitocondrial pyruvate transporter is 0,3 mM [44]Nd: not determined.



dogfish (Squalus acanthias) Ldh protein. This enzyme wasselected because the spatial structure of the enzyme,which is required for the docking simulations, is availablein literature [21]. Because of the high sequence similaritywith Ldh proteins from other sources, simulations datacan be extended to the other enzymes of this family. Thecomparison between the X-ray structures of the apo andholo form of Ldh from S. acanthias reveals that the ami-noacid Asn108 could play a relevant role in the dynamicbehaviour of the catalytic loop. The effect of Asn108replacement on the potential energy profile related to theconformational rearrangement of the catalytic loop hasbeen evaluated in silico, according to the procedure out-lined in Methods. In particular, the computational inves-tigation of five Ldh mutants in which Asn108 has beenreplaced by Asp, Ala, Gly, Lys or His suggests that theAsn108/Gly replacement should lower the energy barrierof the rate determining step, possibly leading to a catalyt-ically more efficient Ldh. In light of these computationalresults, we produced a mutated L. planctarum Ldh wherethe aminoacid Asp94, (corresponding to Asn108 of the S.acanthias enzyme), is replaced by Gly.

Initially we cloned the new LDH gene into an integrativeexpression vector under the control of the constitutive

ScTPI promoter (Table 1). The GRF18U S. cerevisiae strainhas been transformed. Lactate production from the newLdh and the wild-type L. plantarum Ldh (i.e., the control)enzymes were compared, Figure 1A, B. The yeast clonesobtained after transformation with the new LDH geneyielded a much higher lactate accumulation (11300against 6150 mg l-1) and productivity (240 mg l-1 h-1

against 90 mg l-1 h-1 as determined during the exponentialgrowth phase).

Additionally, comparable results have been obtainedtransforming the host cells with two identical centromericplasmids bearing the wild-type (i.e., control) or themutated LDH gene under the control of the same ScTPIpromoter (data not shown).

In principle, the improved production level and produc-tivity rate could be at least associated with a better tran-scription rate of the mutated LDH gene, and/or to a lowerturnover rate of the transcript, and/or to a better transla-tion rate of the transcript and/or to a lower turnover rateof the protein. Since Ldh antibodies are not commerciallyavailable, to address these general considerations, wedeveloped (see MM section) a polyclonal antibodyagainst the L. plantarum Ldh protein. The amounts of the

Wild type Vs mutated LdhFigure 1Wild type Vs mutated Ldh. Recombinant S. cerevisiae GRF18U cells expressing the wild type (closed symbols) or mutated (open symbols) L. plantarum LDH genes were shake-flask grown in glucose 2% wv-1 minimal selective medium till stationary phase. Samples were collected at indicated times for optical density (OD 660 nm, circles; panel A), lactate (g l-1, squares; panel A), ethanol (g l-1, circles; panel B) and residual glucose (g l-1, squares; panel B) determinations.



wild type and mutated proteins were evaluated along thegrowth curves of the recombinant hosts. We also deter-mined the Ldh specific activities. On one side, Figure 2clearly indicates that the amounts of the wild-type andmutated Ldh protein synthesized by the transformedGRF18U strain are similar. On the other side, the specificactivity and the deduced Km value (calculated on crudecell extracts), of the mutated enzyme are different fromthose determined for the wild-type one, being 9,9 U mg-1

and 1,01 mM against 3,2 U mg-1 and 1,504 mM, respec-tively. The higher activity and the lower Km justify thehigher lactate accumulation and the consequently muchhigher yield on glucose: 0,525 against 0,3075 grams oflactate produced per gram of glucose consumed, respec-tively. Surprisingly, the mutated Ldh did also lead to asimilar ethanol production (Fig. 1A) and to a slightly bet-ter biomass production (Fig. 1A); these findings can beassociated with a higher specific glucose consumption rate(Fig. 1B).

Overexpression of JEN1 in yeast hosts producing different amounts of lactateWild type S. cerevisiae cells do not produce lactic acid;however, lactate can be used as carbon and energy source[22]. Lactic acid can freely diffuse through the membranesonly in its undissociated form. Since yeast cells can growon lactate even at pH values much higher than the pKAvalue of the organic acid (i.e., 3,78), a specific transportershould be involved in the uptake of lactate. In S. cerevisiae,synthesis of a lactate permease takes place after transcrip-tion of the JEN1 gene. Transcription of JEN1 is induced bylactate and it is repressed by glucose [23-25]. Jen1 is theonly S. cerevisiae member of the sialate-proton symporters

subfamily belonging to the major facilitator superfamily[26].

It has been already shown that the over-expression ofJEN1 in both S. cerevisiae and Pichia pastoris resulted in anincreased activity of the lactate (inward) transport, whilethe deletion of JEN1 impaired the growth on both lactateand pyruvate [27]. A copy of JEN1 under the control of theconstitutive GAPDH (glyceraldehyde-3-phosphate dehy-drogenase) promoter has been introduced in S. cerevisiaecells using a centromeric plasmid; independently of themechanisms of repression and degradation previouslyreported for the JEN1 gene and Jen1 protein, the cloningunder a strong promoter allowed the constitutive expres-sion of the JEN1 gene even in the presence of glucose [27].

Theoretically, higher lactate productions could beobtained by facilitating the lactate export. In fact, since thecytoplasmic pH value in yeast cells is much higher thanthe lactic acid pKa value, almost all of the lactic acid pro-duced is in the dissociated form and has to be activelytransported outside the cells. A limitation of lactic acidtransport will inevitably lead to an increase in the cyto-plasmic lactate concentration, inhibiting the "in vivo" Ldhactivity and leading to the reduction or arrest of the lactateproduction. Indeed, Figure 3 proves that "in vitro" a lac-tate concentration as low as 10 g l-1 reduces the overall Ldhactivity by about 40%. It can be speculated that the Jen1permease could transport lactate/H+ across the two sidesof the plasma membranes depending on their concentra-tions.

Comparison of wild type and mutated L. plantarum Ldh protein levels: Western Blot determinationFigure 2Comparison of wild type and mutated L. plantarum Ldh protein levels: Western Blot determination. S. cerevisiae GRF18U cells transformed with the plasmid carrying the wild type or the mutated L. plantarum LDH genes were shake-flask grown in glucose 2% wv-1 minimal selective medium. From samples collected at indicated times total protein were TCA extracted and Ldh(s) levels were visualised by Western Blot analyses. Protein extracts corresponding to 107 cells were loaded in each lane, together with a negative control (-).



Based on these considerations and looking to increase lac-tate export and production, we co-expressed the JEN1 andLDH genes in S. cerevisiae host cells growing on 2% glu-cose-YNB. The first findings obtained for the GRF18Uyeast strain transformed with the bovine, the L. casei or theL. plantarum LDH genes showed that the co-expression ofJEN1 was associated with an increase of both lactate pro-duction and yield. An example of such an improvement isshown in Figure 4. Results reported in the figure seem toindicate that the extracellular accumulation of the pro-duced lactate could be modulated by the over-expressionof the Jen1 permease. To test this hypothesis, we checkedthe lactate production from yeast host transformed with aLDH gene and deleted of the endogenous JEN1 gene.When we compared the lactate production from wild typeand jen1 strains transformed with a LDH gene, no differ-ences were observed (data not shown). Since lactate cannot diffuse through the membranes, this simple observa-tion indicates that at least another lactate transporter mustbe operative.

To further investigate the effect of the co-over-expressionof LDH and of the JEN1 genes, we grew different recom-binant yeast strains on glucose 5%-YNB. Essentially, anincrease of the carbon source availability (from 2 to 5%wv-1) simply leads to an increased lactate production. Fig-ure 5 reports the improvements associated to the over-expression of JEN1 against the total lactic acid productionfrom different yeast strains transformed with the L. caseior the L. plantarum LDH genes. The improvement value,for any test, is the ratio between the amount of lactate pro-duced by the yeast strain co-over-expressing JEN1 andLDH and the same strain expressing only the LDH gene(i.e., control). A value of 1 means that an identical lactateproduction was observed, a value of 1.5 means a 50%higher production, a value of 2 means a double produc-tion, and so on. From the analysis of the data it can be eas-ily evinced that co-over-expression of JEN1 has a cleareffect when the production of lactate is very low. How-ever, the effect of Jen1 permease becomes undetectablewhen the lactate production approaches about 8–10 g l-1,suggesting a possible saturation of the Jen1 transportmechanism.

In this respect, we did not try to further over-express theJEN1 gene because it has been shown that its over-expres-sion using a multicopy plasmid did lead to an impairedgrowth [28].

It has been recently suggested that the export of lactatefrom recombinant yeast is an ATP-dependent process[10]. This extra energy required for the export of lactatecould justify the lower biomass yield we generallyobserved for host cells producing a higher amount of lac-tate; Figure 4 for example compares the biomass valuesreached by two strains producing different amounts oflactate, being 2,4 (OD660) for the strain transformed withthe L. plantarum LDH or 2,1 (OD660) for the same straintransformed with the same LDH gene and the S. cerevisiaeJEN1.

In this respect, it is important to underline that an oppo-site behaviour can be obtained following the expressionof the mutated L. plantarum LDH gene. In fact, in this casewe observed a better lactic acid production/productivityand a better biomass production, being 2,4 (OD660) forthe strain transformed with the L. plantarum LDH or 2,6(OD660) the same host strain, but transformed with themutated LDH gene. However, it should be also noted thata similar ethanol production and a higher glucose con-sumption rate have been obtained too. More investiga-tions are required to better understand the biologicalbackground of this behaviour.

"In vitro" inhibition of the Ldh activity by the substrate (lac-tate)Figure 3"In vitro" inhibition of the Ldh activity by the sub-strate (lactate). Total cell proteins were extracted from GRF18U hosts cells expressing the bovine LDH gene. The same amount of protein extract was used to determine the Ldh activity in presence of different amounts of lactate (abscissa) and referred as percentage (ordinate) of the Ldh activity determined in absence of lactate (control).



ConclusionEvolution has produced a huge variety of micro-organ-isms living in radically different environments. In particu-lar, some of these micro-organisms have evolvedmetabolic pathways leading to the synthesis of potentiallyuseful compounds that are difficult to produce from thechemical industry or that are environmentally harmful tomanufacture. Generally speaking, the fundamental basisof evolution is the need to survive and reproduce, not toproduce potentially important and commercially valuableproducts. Indeed, interesting metabolites are very oftenproduced from wild type micro-organisms in such lowconcentrations that biotechnological exploitation isimpractical. The ability to select mutants or to developorganisms by means of rDNA technologies to enable fastand environmentally friendly productions of these prod-ucts has the obvious potential to revolutionize the bio-technological industry. The basic idea of metabolicengineering is to increase the flux through the selectedmetabolic pathway by (i) deleting (where possible) the

competing branch pathways leading to the accumulationof by-products and/or by (ii) over-expressing the proteinscatalysing rate-limiting steps and thereby removing bot-tlenecks. However, metabolic control analysis (MCA) the-ory teaches that, under stationary conditions, thesebottlenecks do not exist. Generally speaking, all of theenzymes along a pathway are more or less equally rate-limiting [29].

As already underlined, not only for the production ofimportant metabolites, but also for a better understandingof the fundamentals of cell biology, detailed studies arerequired. In this respect, we tried to modulate the lactateproduction and yield by improving the efficiency of thelast (i.e., lactate transport) and the penultimate (i.e., Ldhactivity) steps of the pathway leading to the accumulationof lactate from pyruvate. In fact, MCA also anticipates thata bottleneck situation sometimes could hold true forenzymes at the beginning and/or at the end of a pathway,i.e., the steps controlling the starting of the pathway andthe removal of the final product [29].

A yield as high as 0,52 g g-1 can be reached forcing the met-abolic flux towards the accumulation of the final productwithout any disruption and/or limitation of the otherpathways competing for the same substrate. Figure 6,summarizes the production and yield values that can beachieved from the same S. cerevisiae strain (GRF18U)bearing different LDH growing under the same physiolog-ical conditions on 2% wv-1 glucose-YNB.

Concluding, in this manuscript we proved that a highredirection of the glycolytic flux can be also obtained bymodulating the last and the second last steps of the path-way leading to the extracellular accumulation of lactatefrom glucose. In this respect, and to the best of our knowl-edge, higher redirections of the glycolityc flux have neverbeen obtained before without any disruption and/or lim-itation of the competing pathways.

MethodsYeast strains, media and transformationThe S. cerevisiae strains used in this study were GRF18U[30] (MATα; ura3; leu2-3,112; his3-11,15; cir+), CENPK113-11C (MATa ura3-52; his3-11,15;cir+) (see, as an exam-ple [31]), W303-1A (MATa; ade2-1; ura3-1; leu2-3, 112;his3-11, 15; trp1-1; can1-100) [32] and MB11 (MATa;ade2-101; ura3-52; lys2-801; his3-∆200; trp1-∆1; can1)[33].

Yeast cultures were shake-flask grown in minimal syn-thetic medium (0.67% wv-1 YNB Biolife without aminoacids) with 2% wv-1 of glucose as carbon source. Whenrequired, supplements such as leucine, uracil and histi-dine were added to a final concentration of 50 mg l-1. The

Effect of JEN1 expression on lactate production in recom-binant GRF18U host cellsFigure 4Effect of JEN1 expression on lactate production in recombinant GRF18U host cells. S. cerevisiae GRF18U cells transformed with the L. plantarum LDH (control; closed symbols) or co-transformed with the L. plantarum LDH and the endogenous JEN1 genes (open symbols) were shake-flask grown in glucose 2% wv-1 minimal selective medium till sta-tionary phase. Samples were collected at indicated times (h) for optical density (OD 660 nm, circles) and lactate (g l-1, squares) determinations.



cultures were repeated independently (at least) threetimes, samples were taken at different times for the deter-mination of growth parameters, metabolite production,nutrient consumption, enzymatic activities and proteinlevel.

Transformation of S. cerevisiae strains was performedaccording to the LiAc/PEG/ss-DNA protocol [34]. Theyeasts were transformed with one or more of the con-structs described below, together with the respectiveempty plasmid(s). For each set of transformation, inde-pendent clones (at least 4) were initially tested and lactatelevels determined, showing no significative differencesamong the independent clones.

Genes amplification, mutagenesis and expression plasmid constructionFor the expression of the different heterologous LDHactivities and for the over-expression of the S. cerevisiaelactate transporter Jen1, yeast expression plasmids of theseries named "pYX" (R&D Systems Wiesbaden, Germany)have been used. In these plasmids, the cassette for heter-ologous protein expression is based on the constitutive S.cerevisiae TPI promoter and the respective polyA, inter-spaced by the multi-cloning site (MCS), exploited for thesub-cloning of the genes of interest. The selection is basedon different S. cerevisiae auxotrophic markers (in thepresent study always indicated in parenthesis, see alsoTable 1). In particular, the series of plasmids here utilisedare all integrative, and the efficiency of integration wasoptimised by linearizing the plasmids before the yeasttransformation, exploiting a unique site present in thesequence of the auxotrophic marker.

The full length Bos taurus (bovine) LDH-A codingsequence was PCR amplified from the plasmid pLDH12[35] and inserted in the pALTER-1 (Promega) vector asalready described [36]. From the resulting plasmid pVC1the bovine LDH was sub-cloned into the commerciallyavailable yeast integrative vector pYX012 (URA3 marker)generating the expression plasmid pB1. Alternatively, thebovine LDH gene was sub-cloned into the integrative plas-mid pYX022 (HIS3 marker) and pYX042 (LEU2 marker),generating the plasmids pB2 and pB3, respectively. For thedescribed expression plasmids, the enzyme codingsequences were all EcoRI/SalI excised, and sub-cloned inthe receiving expression vectors equally opened.

The Bacillus megaterium and Bacillus stearothermophylusLDH genes (sequences available at the accession n.M22305 and M19396 GenBAnk, respectively) were PCRamplified from genomic DNA of the respective strainsobtained by using the Invisorb Spin Bacteria DNA MiniKit (Invitek), following manufacturer's instructions. Thefollowing primers were designed and used: LDHBM fw: 5'ACA AAT GAA AAC ACA ATT TAC ACC AAA AAC 3'LDHBM rev: 5' ATG TTA CAC AAA AGC TCT GTC GC 3';LDHBSt fw: 5' AAT GAA AAA CAA CGG TGG AGC CCG 3'LDHBSt rev: 5' tgc ctc atc gcg taa aag cac gg 3'. The respec-tive unique fragments obtained were sub-cloned in thevector pSTblue-1 utilising the Perfectly Blunt® Cloning Kit(Novagene) and checked by sequencing analysis. Fromthe obtained plasmids pSTBMLDH and pSTBSTLDH, thecoding sequences were EcoRI excided and subsequentlysub-cloned into the S. cerevisiae integrative expression vec-tor pYX012 (URA3 marker), EcoRI opened and de-phos-phorylated. The resulting expression plasmids werenamed: pBME2 and pBST2, respectively.

JEN1 effect decreases for high lactate productionsFigure 5JEN1 effect decreases for high lactate productions. Different yeast strains (see also Table 2) transformed with the L. casei or L. plantarum LDH genes (i.e., controls) or co-transformed with the same LDH and the S. cerevisiae JEN1 genes (see also Table 1) were shake-flask grown in glucose 2% or 5% wv-1 minimal selective medium till stationary phase. In abscissa are reported the highest lactate production deter-mined, while in the ordinate are reported the improvements observed. The improvement value represents the ratio between the highest lactate production observed in the strain co-expressing the LDH gene and the Jen1 permease and the same strain expressing only the LDH gene.



The Lactobacillus casei LDH gene was PCR amplified andsubsequently sub-cloned into the yeast integrative vectorpYX012 (URA3 marker) generating the expression plas-mid pLC5, or in pYX022 (HIS3 marker) generating theexpression plasmid pLC7, as previously described [30].

The Lactobacillus plantarum LDH gene (sequence availableat the accession n. X70926, GenBAnk) was PCR amplifiedfrom genomic DNA obtained from the strain ATCC 8014by using the Invisorb Spin Bacteria DNA Mini Kit(Invitek), following manufacturer's instructions. The fol-lowing primers were designed and used: LDH fw: 5' TGACTT ATT ATG TCA AGC AT 3', where a mismatch wasintroduced in order to substitute the bacterial start codonTTG with ATG, correctly recognised by S. cerevisiae, andLDH rev: 5' ATC GTA TGA AAT GAT TAT TTA TT 3'. Theunique fragment obtained was sub-cloned in the vectorpSTblue-1 utilising the Perfectly Blunt® Cloning Kit(Novagene) and sequenced. In the amplified sequencefour bp resulted substituted in respect to the originalsequence, but only two of them generated two conserva-tive aa substitutions (H54D and C302S, respectively).From the obtained and sequenced plasmid pSTplLDH,the coding sequence of plLDH was EcoRI excised and sub-sequently sub-cloned into the S. cerevisiae integrativeexpression vector pYX022 (HIS3 marker), pYX012 (URA3marker) EcoRI opened and de-phosphorylated. The result-ing expression plasmids were named p022TLP andp012TLP, respectively.

The plLDH was then also mutagenised according to thesuggestions derived from modelling studies (see below).In particular, the aa 94 Asp (D) was changed into Gly (G)by a double bp substitution changing the triplet GAC intoGGT. The mutagenesis was made by the Altered Sites II invitro Mutagenesis Systems (Promega) using plasmid pST-plLDH as template DNA and complementary primer pairsthat encode the desired amino acid replacement, follow-ing manufacturer's instructions. The sequence change wasconfirmed by DNA sequence analysis and finally sub-cloned into plasmids of the already described series"pYX". In particular, the modified sequence was insertedin the plasmid pYX022 (HIS3 marker) or in the plasmidpYX012 (URA3 marker), resulting in the yeast expressionplasmids p022TLPD94G and p012TLPD94G, respec-tively.

The DNA sequence of JEN1 (available at the accession n.U24155, GenBank) was PCR amplified with the followingprimers: Tony s: 5' ACT GCT ACT GAA AAT ATG TCG TCGT 3' and Tony as: 5' AGT GAT TAA ACG GTC TCA ATATGC TC 3' starting from S. cerevisiae genomic DNAextracted as described (slightly modified from [37]). Theunique fragment obtained was sub-cloned in the vectorpSTblue-1 and sequenced. From the obtained plasmid,

the coding sequence of ScJEN1 was EcoRI excided and sub-sequently sub-cloned into the S. cerevisiae integrativeexpression vectors pYX012 (URA3 marker) and pYX022(HIS3 marker) EcoRI opened and de-phosphorylated, gen-erating the plasmids p012Jen1 and p022Jen1, respec-tively.

DNA manipulations, transformation and cultivation ofEscherichia coli (DH5αF' (φ 80d lacZ∆M15, ∆ (lacZYA-argF), U169, deo, rec1, end1, sup44, λ, THI-1, gyrA96, relA1and Novablue Competent Cells (Novagene) were per-formed following standard protocols [38]. All the restric-tion and modification enzymes used were from NewEngland Biolabs (Hitchin, Herts, UK) or from RocheDiagnostics (Mannheim, Germany).

Measurement of cell concentration, metabolites and enzymatic activitiesIndependent recombinant yeast transformants wereshake-flasks cultured in minimal medium. During the cul-tures, followed up to stationary phase, samples were col-lected at regular time intervals. Cell concentration wasdetermined by measuring the optical density at 660 nm orthe cell number by a Coulter Counter determination [39].

Glucose, ethanol, L(+)-lactate were determined by usingdiagnostic kits (Boehringer Mannheim cat n° 148261,716251 and 176290, respectively) according to manufac-turer's instructions. The lactate yields were calculated bylinear regressions obtained by plotting the grams of glu-cose consumed in the course of fermentation processesversus the grams of lactate produced.

For all LDH activities, samples were prepared as follows:about 108 cells were harvested, washed in ice-cold waterand re-suspended in 50 mM phosphate buffer pH 7.5,Glycerol 20%, PMSF 1 mM and protease inhibitors (Com-plete EDTA-free Protease inhibitor cocktail tablets,Roche). Cells were broken with 5 cycles of vigorous vor-texing in presence of glass beads (diam. 400–600 µm,Sigma) at 4°C. After centrifugation, protein extracts con-centration was determined (Bradford, Biorad).

For bovine LDH activity, about 0.2 mg of extract weretested using the Sigma kit DG1340-UV, according to man-ufacturer's instructions.

For all bacterial LDH activities determination, except theL. plantarum one, cellular extract (0.05 ml of properlydiluted samples) were incubated with 0.01 ml of 12.8 mMNADH, 0.1 ml of 2 mM fructose1,6-diphosphate, 0.74 mlof 50 mM acetate buffer (pH 5.6) and 0.1 ml sodiumpyruvate 100 mM. The L. plantarum LDH activity wasalmost identically determined, omitting the not necessarycofactor fructose1,6-diphosphate, as previously described



[40]. LDH activity was assayed as micromoles of NADHoxidized per min, per mg of total protein extract, at 340nm, 25°C.

SimulationsThe computational investigation has been carried outusing the X-ray structure of Ldh from Squalus acanthias,chosen because both of the holo and apo structures areavailable (pdb codes: 3LDH, 6LDH, respectively. Website: http://www.rcsb.org/pdb). However, results areexpected to be valid also for Ldh from L. planctarum, dueto the high sequence similarity between the two proteins(not shown).

Molecular Mechanics (MM) optimizations were per-formed using the GROMACS simulation software package[41], implemented on a parallel architecture. MM runs

consisted in 1000 steepest descent cycles followed by10000 conjugate gradient steps.

Initially, cofactor (NADH) and substrate (pyruvate) havebeen removed from the X-ray structure, in order to sim-plify the computational investigation of the conforma-tional properties of the protein. Since the movement ofthe catalytic loop takes place on a relatively low time scale(kcat = 250 s-1), average value among several species [20]standard Molecular Dynamics simulations could not beused to investigate the conformational rearrangement.Instead, the conformational properties of the catalyticloop have been investigated by iterative modification ofselected torsional angles, followed by Molecular Mechan-ics optimization. In particular, analysis of the X-ray struc-tures of the apo and holo form of Ldh suggests that theconformational rearrangement of the catalytic loopmainly implies modification of two protein torsion angles(phi96 and phi97, N-Calfa), which have been systematicallyrotated in steps of 10 degrees. The resulting structureswere optimised by MM in order to obtain stationarypoints on the Potential Energy Surface (PES), thus allow-ing to sample the PES of the protein structure along thereaction coordinate corresponding to the relevant move-ment of the catalytic loop.

Antibody development against L. plantarum LDH and protein level analysisDifferent commercially available antibodies against LDHwere tested, but none of them is specific for bacterialLDHs and none of them resulted sufficiently specific forplLDH detection in our host system. By utilizing the toolSyfpeithi http://www.bmi-heidelberg.com/ we individu-ated the peptide DCKDADLVVITAGAPQKPGE (from aa71 to aa 90 of the aminoacidic sequence of the protein) asthe preferred immunogen to develop polyclonal anti-body. The above mentioned peptide and the correspond-ing antibody (as immune serum) were synthesized anddeveloped, respectively, by Primm Italia http://www.primmbiotech.com.

LDH expression levels were analyzed by Western Blot.From each point of the above described kinetics ofgrowth, a culture volume corresponding to 108 cells washarvested by centrifugation, and crude extracts were pre-pared by following the trichloroacetic acid protocol [42]and resuspending the final protein extract in 150 µl ofLaemmli buffer [43]. Protein extracts corresponding to107 cells were loaded on a 12% polyacrylamide gel (SDS-PAGE); after separation, proteins were blotted to nitrocel-lulose membranes and immunodecorated. LDH levelswere detected using the developed primary rabbit polyclo-nal antibody, (dilution 1:250) and a secondary anti-rabbitIgG horseradish peroxidase-conjugated (Amersham Phar-macia Biotech, UK; dilution 1:5000) antibody. The

Lactate production and yield obtained from recombinant GRF18U host cellsFigure 6Lactate production and yield obtained from recom-binant GRF18U host cells. Different recombinant S. cere-visiae GRF18U cells were shake-flask grown in glucose 2% wv-1 minimal selective medium till stationary phase. Samples were collected at indicated times (h) for lactate (g l-1) and yield (gram of lactate produced per gram of glucose con-

sumed; values are here below reported) determinations. ( ) B. taurus LDH yield 4% ± 0.2% (�) L. casei LDH yield 21% ± 0.4% (■) L. plantarum LDH yield 31% ± 0.5% (�) L. plantarum LDH + ScJEN1 yield 39% ± 0.5% (●) Mutated L. plantarum LDH. yield 52% ± 0.7%


http://www.rcsb.org/pdbhttp://www.bmi-heidelberg.com/http://www.primmbiotech.comhttp://www.primmbiotech.com


immunoreactive protein bands were developed using theSuper Signal West Pico Western blotting system (Pierce),according to the manufacturer's instructions.

Authors' contributionsPB participated in the design of the study and plannedand carried out the experimental work including plasmidand strain construction, yeast cultivation and data analy-sis, western blot immunoassays and enzymatic activitydetermination; participated in drafting the manuscript.

MS and MV participated in the design of the study and inmolecular biology experiments.

LDG and GZ designed and performed the modeling stud-ies and simulations, suggesting the point mutation.

DM participated in the design of this study and in datainterpretation.

DP designed the whole study, performed data interpreta-tion and drafted the manuscript.

All authors read and approved the final manuscript.

AcknowledgementsThis work was partially supported by Tate & Lyle North America, Decatur, Illinois and F.A.R. 2005 to DP. The work related to the mutated LDH was partially supported by the Project BIOCDM (Biotech Clusters Develop-ment and Management) Interreg IIIC – REGINS, 2005 to DP. The authors are indebted to Marina Vai, Vittorio Carrera and Luca Brambilla for helping with some of the presented cloning work and experiments and to Carla Smeraldi for language corrections throughout the manuscript.

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AbstractBackgroundResultsConclusion

BackgroundResults and discussionExpression of different LDH(s) in different yeast hostsImproving lactate production and yield by protein engineeringOverexpression of JEN1 in yeast hosts producing different amounts of lactate

ConclusionMethodsYeast strains, media and transformationGenes amplification, mutagenesis and expression plasmid constructionMeasurement of cell concentration, metabolites and enzymatic activitiesSimulationsAntibody development against L. plantarum LDH and protein level analysis

Authors' contributionsAcknowledgementsReferences

Documents

Microbial Cell Factories BioMed CentralLdh(s) under the control of the same ScTPI promoter (data not shown). Improving lactate production and yield by protein engineering The Ldh enzyme